1. Field of Invention
This application relates to microscopes and methods of microscopy, and more particularly to multi-photon microscopes and multi-photon methods of microscopy.
2. Discussion of Related Art
The contents of all references, including articles, published patent applications and patents referred to anywhere in this specification are hereby incorporated by reference.
Laser fluorescence confocal microscopy is an effective technique for producing three-dimensional images. Typically, the optical sectioning is achieved by placing a pinhole aperture in front of the emission detector. Alternatively, multi-photon fluorescence excitation microscopy (MPFM) techniques (two-photon, three-photon, second harmonic generation, sum frequency generation, etc.) can be used to provide optical sectioning by limiting fluorescence excitation to a point source in the focal plane. The technique of two-photon microscopy was introduced by Denk et al. in “Two-Photon Laser Scanning Fluorescence Microscopy”, Science, Vol. 248, pp. 73-76, (April, 1990) (see also Denk et al. in U.S. Pat. No. 5,034,613). Two-photon fluorescence microscopy (TPFM) has advantages that include that it causes less damage to the biological system above and below the focal plane and that longer excitation wavelengths can be used to excite fluorescence from deeper in a sample (hundreds of microns).
In MPFM, the excitation is limited to the focal plane due to the level of spatial and temporal crowding of photons into diffraction limited spot. This crowding increases the probability of a fluorophore absorbing multiple photons before relaxation to the ground state or it increases the probability of coherent scattering events. In the case of (TPFM) in which two photons are of the same wavelength, the excited state is at twice the energy of the photons used for excitation. Since multi-photon absorption is a lower probability event than single photon absorption, a high intensity illumination source is typically required to excite a sufficient number of molecules to be detected. Once the multi-photon excitation condition is met emission light propagates in all directions from the excited spot. Since there is no need for using a pinhole aperture for optical selection the opportunity for collecting all of the light, regardless of the direction of propagation, exists for optimizing light collection. Conventional multi-photon microscopes illuminate and collect light through the same objective lens system or in conjunction with a detector placed in the trans-fluorescence pathway. This leads to detecting only a fraction of the light that is emitted from the sample. More light collection means less excitation power is needed and deeper tissue penetration is possible. Thus, there is thus a need for multi-photon microscopes that can obtain improved light collection emitted from an object being observed for a given illumination intensity.